The Apoptosis Induced by Ceramide in the Endothelial Cell.

Lee, Sun Young; Kim, Tae Im; Tchag, Hungwon

Original Article

Journal of the Korean Ophthalmological Society 2003;44(9):2128-2136.
Published online September 1, 2003.

The Apoptosis Induced by Ceramide in the Endothelial Cell.

Sun Young Lee, Tae Im Kim, Hungwon Tchag

Department of Ophthalmology, College of Medicine, University of Ulsan, Asan Medical Center, Korea. hwtchah@amc.seoul.kr

각막내피세포에서 Ceramide에 의해 유발된 Apoptosis

이선영 ( Lee Seon Yeong ) , 김태임 ( Kim Tae Im ) , 차흥원 ( Cha Heung Won )

Abstract

PURPOSE
To evaluate the effect of variable ceramides on the apoptosis of corneal endothelial cell and then, if ceramide induce the apoptosis in endothelial cells, via which pathway apoptosis occur. METHODS: Corneal endothelial cells were isolated from fresh rabbit cornea and cultured. Cultured corneal endothelial cells were exposed to 10, 20, 40 and 80 micro M of ceramide type II, VI and phytoceramide type II, VI. And then, apoptosis was evaluated with Hoechst staining and flow cytometric analysis with Annexin V for evaluation of apoptotic response. Corneal endothelial cells were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK(R)), specific caspase-8 inhibitor(IETD-CHO(R)) and specific caspase-9 inhibitor (Z-LEHD-FMK(R)), then treated with 20 M of 4 types of ceramide. 12 hours later, LDH assay was done. Cytochrome c immunostaining was done after exposure to 4 types of ceramide. RESULTS: Shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation were observed on Hoechst staining. In flow cytometric analysis, early apoptotic responses were identified. Apoptotic response increased significantly at the concentration of 10M and more 12 hours later. CPP32-like protease inhibitor, caspase-8, 9 inhibitor reduced the LDH activity. Apoptotic endothelial cells induced by ceramide were stained with cytochrome c antibody. CONCLUSIONS: Ceramide induced apoptosis in cultured corneal endothelial cells. This apoptosis developed via caspase and mitochondrial pathway.

Key Words: Apoptosis;Caspase;Ceramide;Corneal endothelial cell;Mitochondrial pathway;Phytoceramide